The methylation status of DNA influences many biological processes during mammalian development, and is known to be highly aberrant in cancer. In mammalian cells, DNA methylation occurs as symmetrical methylation of cytosine in the context of the dinucleotide CpG, and the presence of high levels of 5-methylcytosine (5-mC) are generally correlated with diminished gene expression. We recently discovered that the TET proteins TET1, TET2 and TET3 constitute a new family of 1- ketoglutarate (1KG)- and Fe(II)-dependent dioxygenases that catalyse the hydroxylation of 5- methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC, TET1 and TET2 are present at high levels in DNA of mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, suggesting potential roles in pluripotency. Moreover, 5-hmC levels and TET expression/ activity are tightly regulated: hmC is present in genomic DNA of undifferentiated ES cells but not several differentiated cell types, and hmC levels diminish upon ES cell differentiation. Together these data suggest that dysregulation of DNA methylation via TET proteins and hmC may have a role in ES cell pluripotency. Here we propose to analyze the biological roles of Tet proteins in gene expression, pluripotency and cell fate specification in mouse ES and iPS cells. In Aim 1, we will study the roles of Tet proteins in mouse ES cell pluripotency and differentiation. In Aim 2, we will examine the requirement for Tet proteins in reprogramming murine fibroblasts to iPS cells. In Aim 3, we will explore the roles of 5-hmC and Tet proteins in gene expression in ES cells. The results should provide new insights into the role of the novel base, 5-hmC, and the recently-discovered TET family of enzymes, in maintenance of the pluripotent state in ES and iPS cells. PUBLIC HEALTH RELEVANCE: In addition to the four major bases in the DNA alphabet - A, C, G and T - there is also a very minor base known as 5-methylcytosine (5mC) that has a disproportionately crucial role. This base is produced from the major base cytosine (C) by attaching a methyl group to its "5" position. Interference with cytosine methylation can lead to a number of developmental abnormalities, genetic diseases and cancer. We recently identified a new class of proteins known as TET proteins that convert 5- methylcytosine to a variant known as 5-hydroxymethylcytosine (hmC). TET proteins and 5-hmC are are strongly expressed in mouse embryonic stem (ES) cells, and are induced to high levels when mouse fibroblasts are reprogrammed into induced pluripotent stem (iPS) cells. In this proposal we plan to investigate the role of TET1 and TET2 proteins in maintaining the pluripotent state of ES cells and iPS cells.